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United States Patent |
6,262,263
|
Pennington
,   et al.
|
July 17, 2001
|
Quinolinium- and pyridinium-based fluorescent dye compounds
Abstract
The present invention relates to quinolinium- and pyridinium-based
fluorescent dyes. The dyes are useful for staining proteins in solution,
in gels and on solid supports. The dyes of the invention exhibit higher
fluorescence emission than known compounds when bound to proteins and also
exhibit improved contrast of fluorescence intensity between their
protein-bound and unbound states.
Inventors:
|
Pennington; Mark William (Oxford, GB);
Scopes; David Ian (Tubney, GB);
Orchard; Michael Glen (Watlington, GB)
|
Assignee:
|
Oxford Glycosciences (UK) Ltd. (Abingoon, GB)
|
Appl. No.:
|
412168 |
Filed:
|
October 5, 1999 |
Foreign Application Priority Data
| Oct 05, 1998[GB] | 9821682 |
| Oct 05, 1998[GB] | 9821683 |
| Oct 05, 1998[GB] | 9821684 |
Intern'l Class: |
C07D 401/10; C07D 403/10; C07D 215/12; C07D 213/36; C07D 213/57 |
Field of Search: |
544/359,363,124,128
546/330,334,172
|
References Cited
U.S. Patent Documents
5616502 | Apr., 1997 | Haugland | 436/86.
|
5912257 | Jun., 1999 | Prasad | 514/356.
|
Foreign Patent Documents |
0 548 798 | Jun., 1993 | EP.
| |
WO 96/36882 | Nov., 1996 | WO.
| |
WO 98/23950 | Jun., 1998 | WO.
| |
Other References
Basili, 1997, "Development of a High-Throughput Fluorescence Scanner
Employing Internal Reflection Optics and Phase-Sensitive Detection",
Dissertation Abstracts 58(12-B):6686 (Ph.D. Thesis, University of
Washington).
|
Primary Examiner: Huang; Evelyn Mei
Attorney, Agent or Firm: Pennie & Edmonds LLP
Claims
What is claimed is:
1. A compound of formula I
##STR35##
wherein
R.sup.1 is (C.sub.1 -C.sub.6) straight or branched chain alkyl, halogen or
--CF.sub.3 ;
either R.sup.2a and R.sup.2b are independently (C.sub.1 -C.sub.20) straight
or branched chain alkyl, (C.sub.1 -C.sub.20) straight or branched chain
aralkyl or H, R.sup.2a and R.sup.2b not simultaneously being H, or R.sup.2
a and R.sup.2 b are taken together and form a morpholinyl, piperidinyl or
pyrrolidinyl ring;
R.sup.3 is H or (C.sub.1 -C.sub.6) straight or branched chain alkyl; and
either R.sup.4 and R.sup.5 are both H, or R.sup.4 and R.sup.5 taken
together are --CH.dbd.CH--CH.dbd.CH--,
the aromatic rings A and B, the --(CH.sub.2).sub.4-5 -- group, and the
--C(H).dbd.C(R.sup.3)-- group being optionally substituted with one or
more --OH, (C.sub.1 -C.sub.6) straight or branched chain alkoxyl, halogen,
(C.sub.1 -C.sub.6) straight or branched chain haloalkyl, pyridyl,
thiophenyl, furyl, and phenyl, the phenyl being optionally substituted
with one or more --OH, (C.sub.1 -C.sub.6) straight or branched chain alkyl
or (C.sub.1 -C.sub.6) straight or branched chain alkoxyl.
2. The compound of claim 1, selected from the group consisting of:
Quinolinium,
4-[2-[4-(dipentylamino)-3-chlorophenyl]ethenyl]-1-(sulfobutyl)-, inner
salt;
Quinolinium,
4-[2-[4-(dipentylamino)-3-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-,
inner salt;
Quinolinium,
4-[2-[4-(dipentylamino)-3-methylphenyl]ethenyl]-1-(sulfobutyl)-, inner
salt;
Quinolinium,
4-[2-[4-(decylamino)-3-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-,
inner salt; and
Pyridinium,
4-[2-[4-(dipentylamino)-3-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-,
inner salt.
3. The compound of claim 1, selected from the group consisting of:
Quinolinium,
4-[2-[4-(dipentylamino)-3-trifluoromethylphenyl]ethenyl]-1(sulfobutyl)-,
inner salt;
Quinolinium,
4-[2-[4-(dipentylamino)-3-chlorophenyl]ethenyl]-1-(sulfobutyl)-, inner
salt;
Quinolinium,
4-[2-[4-(decylamino)-3-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-,
inner salt; and
Pyridinium,
4-[2-[4-(dipentylamino)-3-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-,
inner salt.
4. A compound of formula I
##STR36##
wherein
R.sup.1 is H, (C.sub.1 -C.sub.6) straight or branched chain alkyl, halogen
or --CF.sub.3 ;
R.sup.2a and R.sup.2b are taken together to form --(CH.sub.2).sub.2
--NR.sup.6 --(CH.sub.2).sub.2 --, wherein R.sup.6 is (C.sub.1 -C.sub.12)
straight or branched chain alkyl, (C.sub.1 -C.sub.12) straight or branched
chain alkylcarbonyl or (C.sub.1 -C.sub.12) straight or branched chain
alkylsulphonyl;
R.sup.3 is H; and
either R.sup.4 and R.sup.5 are both H, or R.sup.4 and R.sup.5 are taken
together and form --CH.dbd.CH--CH.dbd.CH--,
the aromatic rings A and B, the --(CH.sub.2).sub.4-5 -- group, and the
--C(H).dbd.C(R.sup.3)-- group being optionally substituted with one or
more --OH, (C.sub.1 -C.sub.6) straight or branched chain alkoxyl, halogen,
(C.sub.1 -C.sub.6) straight or branched chain haloalkyl, pyridyl,
thiophenyl, furyl, and phenyl, the phenyl being optionally substituted
with one or more --OH, (C.sub.1 -C.sub.6) straight or branched chain alkyl
or (C.sub.1 -C.sub.6) straight or branched chain alkoxyl.
5. The compound of claim 4, selected from the group consisting of:
Quinolinium,
4-[2-[4-[(1-oxooctyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt;
Quinolinium,
4-[2-[4-[(1-oxodecyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt;
Pyridinium,
4-[2-[4-[(1-oxooctyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt;
Pyridinium,
4-[2-[4-[(1-oxodecyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt;
Pyridinium,
2-[2-[4-[(1-oxooctyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt; and
Pyridinium,
2-[2-[4-[(1-oxodecyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt.
6. A compound of formula I
##STR37##
wherein
R.sup.1 is H, (C.sub.1 -C.sub.6) straight or branched chain alkyl, halogen,
or --CF.sub.3 ;
either R.sup.2a and R.sup.2b are independently a lipophilic group or H,
R.sup.2a and R.sup.2b not simultaneously being H, or R.sup.2a and R.sup.2b
are taken together and form a morpholinyl, piperidinyl, pyrrolidinyl, or
piperazinyl ring, wherein the piperazinyl ring is optionally substituted
with (C.sub.1 -C.sub.12) straight or branched chain alkyl, (C.sub.1
-C.sub.12) straight or branched chain alkylcarbonyl or (C.sub.1 -C.sub.12)
straight or branched chain alkylsulphonyl;
R.sup.3 is --CN, CONH.sub.2, --COOH, or --COOR, wherein R is (C.sub.1
-C.sub.6) straight or branched chain alkyl or (C.sub.1 -C.sub.10) straight
or branched chain aralkyl, the --CONH.sub.2 group being optionally
substituted with one or two (C.sub.1 -C.sub.6) alkyl groups or one or two
(C.sub.1 -C.sub.10) aralkyl groups; and
either R.sup.4 and R.sup.5 are both H, or R.sup.4 and R.sup.5 are taken
together and form --CH.dbd.CH--CH.dbd.CH--,
the aromatic rings A and B, the --(CH.sub.2).sub.4-5 -- group, and the
--C(H).dbd.C(R.sup.3)-- group being optionally substituted with one or
more --OH, (C.sub.1 -C.sub.6) straight or branched chain alkoxyl, halogen,
(C.sub.1 -C.sub.6) straight or branched chain haloalkyl, pyridyl,
thiophenyl, furyl, and phenyl, the phenyl being optionally substituted
with one or more --OH, (C.sub.1 -C.sub.6) straight or branched chain
alkyl, or (C.sub.1 -C.sub.6) straight or branched chain alkoxyl.
7. The compound of claim 6, wherein R.sup.3 is --CN.
8. The compound of claim 6, selected from the group consisting of:
Pyridinium, 4-[2-[4-(dibutylamino)phenyl]-1-cyanoethenyl]-1-(sulfobutyl)-,
inner salt; and
Pyridinium, 4-[2-[4-(dihexylamino)phenyl]-1-cyanoethenyl]-1-(sulfobutyl)-,
inner salt.
Description
1. FIELD OF THE INVENTION
The present invention relates to quinolinium- and pyridinium-based
fluorescent dyes, and to their use in the staining of proteins in
solution, in gels and on solid supports.
2. BACKGROUND OF THE INVENTION
Structures of formula II are disclosed in WO 96/36882, which discloses
unsubstituted compounds (R1=R3=H) and the corresponding 2- and
2,6-substituted anilines, and those where the olefinic bond is optionally
substituted (R4, R5=H, alkyl or phenyl), having fluorescence properties. A
specific example of such a compound is shown in formula III.
Structures of formula IV are also disclosed in WO 96/36882, which discloses
unsubstituted compounds (R1=R3=H) and the 2- and 2,6-substituted anilines,
having fluorescence properties. A specific example of such a compound is
shown in formula V.
##STR1##
##STR2##
Citation of a reference herein shall not be construed as indicating that
such reference is prior art to the present invention.
3. SUMMARY OF THE INVENTION
In a first aspect, the present invention relates to compounds of formula I
##STR3##
wherein
R.sup.1 is (C.sub.1 -C.sub.6) straight or branched chain alkyl, halogen or
--CF.sub.3 ;
either R.sup.2a and R.sup.2b are independently a lipophilic group or H,
R.sup.2a and R.sup.2b not simultaneously being H, or R.sup.2a and R.sup.2b
are taken together and form a morpholinyl, piperidinyl or pyrrolidinyl
ring;
R.sup.3 is H or (C.sub.1 -C.sub.6) straight or branched chain alkyl; and
either R.sup.4 and R.sup.5 are both H, or R.sup.4 and R.sup.5 taken
together are --CH.dbd.CH--CH.dbd.CH--,
the aromatic rings A and B, the --(CH.sub.2).sub.4-5 -- group, and the
--C(H).dbd.C(R.sup.3)-- group being optionally substituted with one or
more --OH, (C.sub.1 -C.sub.6) straight or branched chain alkoxyl, halogen,
(C.sub.1 -C.sub.6) straight or branched chain haloalkyl, pyridyl,
thiophenyl, furyl, and phenyl, the phenyl being optionally substituted
with one or more --OH, (C.sub.1 -C.sub.6) straight or branched chain alkyl
or (C.sub.1 -C.sub.6) straight or branched chain alkoxyl.
In a second aspect, the present invention relates to compounds of formula I
wherein
R.sup.1 is H, (C.sub.1 -C.sub.6) straight or branched chain alkyl, halogen
or --CF.sub.3 ;
R.sup.2a and R.sup.2b are taken together to form --(CH.sub.2).sub.2
--NR.sup.6 --(CH.sub.2).sub.2 --, wherein R.sup.6 is (C.sub.1 -C.sub.12)
straight or branched chain alkyl, (C.sub.1 -C.sub.12) straight or branched
chain alkylcarbonyl or (C.sub.1 -C.sub.12) straight or branched chain
alkylsulphonyl;
R.sup.3 is H; and
either R.sup.4 and R.sup.5 are both H, or R.sup.4 and R.sup.5 are taken
together and form --CH.dbd.CH--CH.dbd.CH--,
the aromatic rings A and B, the --(CH.sub.2).sub.4-5 -- group, and the
--C(H).dbd.C(R.sup.3)-- group being optionally substituted with one or
more --OH, (C.sub.1 -C.sub.6) straight or branched chain alkoxyl, halogen,
(C.sub.1 -C.sub.6) straight or branched chain haloalkyl, pyridyl,
thiophenyl, furyl, and phenyl, the phenyl being optionally substituted
with one or more --OH, (C.sub.1 -C.sub.6) straight or branched chain alkyl
or (C.sub.1 -C.sub.6) straight or branched chain alkoxyl.
In a third aspect, the present invention relates to compounds of formula I
wherein
R.sup.1 is H, (C.sub.1 -C.sub.6) straight or branched chain alkyl, halogen,
or --CF.sub.3 ;
either R.sup.2a and R.sup.2b are independently a lipophilic group or H,
R.sup.2a and R.sup.2b not simultaneously being H, or R.sup.2a and R.sup.2b
are taken together and form a morpholinyl, piperidinyl, pyrrolidinyl, or
piperazinyl ring, wherein the piperazinyl ring is optionally substituted
with (C.sub.1 -C.sub.12) straight or branched chain alkyl, (C.sub.1
-C.sub.12) straight or branched chain alkylcarbonyl or (C.sub.1 -C.sub.12)
straight or branched chain alkylsulphonyl;
R.sup.3 is --CN, CONH.sub.2, --COOH, or --COOR, wherein R is (C.sub.1
-C.sub.6) straight or branched chain alkyl or (C.sub.1 -C.sub.10) straight
or branched chain aralkyl, the --CONH.sub.2 group being optionally
substituted with one or two (C.sub.1 -C.sub.6) alkyl groups or one or two
(C.sub.1 -C.sub.10) aralkyl groups; and
either R.sup.4 and R.sup.5 are both H, or R.sup.4 and R.sup.5 are taken
together and form --CH.dbd.CH--CH.dbd.CH--,
the aromatic rings A and B, the --(CH.sub.2).sub.4-5 -- group, and the
--C(H).dbd.C(R.sup.3)-- group being optionally substituted with one or
more --OH, (C.sub.1 -C.sub.6) straight or branched chain alkoxyl, halogen,
(C.sub.1 -C.sub.6) straight or branched chain haloalkyl, pyridyl,
thiophenyl, furyl, and phenyl, the phenyl being optionally substituted
with one or more --OH, (C.sub.1 -C.sub.6) straight or branched chain alkyl
or (C.sub.1 -C.sub.6) straight or branched chain alkoxyl.
The present invention may be understood more fully by reference to the
detailed description and illustrative examples which are intended to
exemplify non-limiting embodiments of the invention.
4. DETAILED DESCRIPTION OF THE INVENTION
4.1 Definitions
As used herein, halogen refers to F, Cl, Br or I.
As used herein, (C.sub.1 -C.sub.6) straight or branched alkyl includes but
is not limited to methyl, ethyl, n-propyl, propan-1-yl, propan-2-yl,
cyclopropan-1-yl, n-butyl, butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl,
2-methyl-propan-2-yl, cyclobutan-1-yl, 3-methyl-n-butyl, n-pentyl,
pentan-1-yl, pentan-2-yl, cyclopentan-1-yl, n-hexyl, hexan-1-yl,
hexan-2-yl, and cyclohexan-1-yl.
As used herein (C.sub.1 -C.sub.6) straight or branched chain alkoxy
includes but is not limited to 2-hydroxyethyl, 2-hydroxypropyl,
3-hydroxypropyl, 4-hydroxybutyl, 1-hydroxybutyl, and 6-hydroxyhexyl.
As used herein, a lipophilic group refers to (C.sub.1 -C.sub.20) straight
or branched chain alkyl or (C.sub.1 -C.sub.20) straight or branched chain
aralkyl.
As used herein, (C.sub.1 -C.sub.20) straight or branched chain alkyl
includes but is not limited to methyl, ethyl, n-propyl, propan-1-yl,
propan-2-yl, cyclopropan-1-yl, n-butyl, butan-1-yl, butan-2-yl,
2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,
3-methyl-n-butyl, n-pentyl, pentan-1-yl, pentan-2-yl, cyclopentan-1-yl,
n-hexyl, hexan-1-yl, hexan-2-yl, and cyclohexan-1-yl, n-octyl, n-decyl,
n-hexadecyl, n-octadecyl, and n-eicosyl.
As used herein, aryl refers to phenyl, naphthyl, or anthracenyl.
As used herein, (C.sub.1 -C.sub.6) straight or branched chain haloalkyl
refers to an alkyl moiety, such as one of those listed above, having at
least one halogen attached, including, but not limited to, --CF.sub.3,
--CH.sub.2 F, --CHF.sub.2, --CH.sub.2 I, --CH.sub.2 Br, --CH.sub.2 Cl,
--CHCl.sub.2, --CCl.sub.3, --CH.sub.2 CHClCH.sub.3, --CH.sub.2
C(CH.sub.3)(CH.sub.2 Br), --CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 I,
--C(CH.sub.3)(CH.sub.3)(CH.sub.2 F), and --CH.sub.2 CHClCH.sub.2
CHClCH.sub.2 CH.sub.3. Preferably, the haloalkyl group is --CF.sub.3.
As used herein, (C.sub.1 -C.sub.12) straight or branched chain
alkylcarbonyl refers to an alkyl moiety, such as those listed above,
having one or more C.dbd.O groups attached.
As used herein, (C.sub.1 -C.sub.12) straight or branched chain
alkylsulphonyl refers to an alkyl moiety, such as those listed above,
having one or more --SO.sub.2 -- groups attached.
One skilled in the art will readily appreciate that when R.sup.4 and
R.sup.5 taken together are --CH.dbd.CH--CH.dbd.CH--, aromatic ring B and
--CH.dbd.CH--CH.dbd.CH-- generate a quinoline system.
4.2 Compounds
The compounds of formula I have been found to display fluorescence
properties superior to those of the prior art. Thus, in particular,
certain compounds of the invention display much increased brightness over
the compounds of formulae III and V when they are bound to protein (e.g.
bovine serum albumin) and when sodium dodecyl sulphate is present in an
amount below its critical micelle concentration. Furthermore, certain
compounds of this invention display greater differences in the
fluorescence intensities of their protein-bound and unbound states when
compared to the compounds of formulae III and V.
Without being bound by any particular theory, Applicants believe that the
compounds of formula I have properties superior to those described in WO
96/36882. In general terms, other substituents (both those shown in
formula I, and also those not shown) may be less critical; thus, each of
the aromatic rings, the --(CH.sub.2).sub.4-5 -- group and the conjugating
C.dbd.C link may carry other/further substituents that do not
substantially affect the fluorescence intensity of the compounds (e.g.
compounds have at least 50%, and preferably at least 75%, of the intensity
of any compound illustrated herein under the same conditions). Examples of
substituents are disclosed in WO 96/36882, the contents of which are
incorporated herein by reference.
Preferred compounds of formula I, of the first aspect of the invention,
include:
Quinolinium,
4-[2-[4-(dipentylamino)-3-chlorophenyl]ethenyl]-1(sulfobutyl)-, inner
salt;
Quinolinium,
4-[2-[4-(dipentylamino)-3-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-,
inner salt;
Quinolinium,
4-[2-[4-(dipentylamino)-3-methylphenyl]ethenyl]-1-(sulfobutyl)-, inner
salt;
Quinolinium,
4-[2-[4-(decylamino)-3-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-,
inner salt; and
Pyridinium,
4-[2-[4-(dipentylamino)-3-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-,
inner salt.
Particularly preferred compounds of formula I, of the first aspect of the
invention, are:
Quinolinium,
4-[2-[4-(dipentylamino)-3-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-,
inner salt;
Quinolinium,
4-[2-[4-(dipentylamino)-3-chlorophenyl]ethenyl]-1-(sulfobutyl)-, inner
salt;
Quinolinium,
4-[2-[4-(decylamino)-3-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-,
inner salt; and
Pyridinium, 4-[2-[4-(dipentylamino)-3-trifluoromethylphenyl]ethenyl]1
(sulfobutyl)-, inner salt.
Preferred compounds of formula I, of the second aspect of the invention,
include:
Quinolinium,
4-[2-[4-[(1-oxooctyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt;
Quinolinium,
4-[2-[4-[(1-oxodecyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt;
Pyridinium,4-[2-[4-[(1-oxooctyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-
inner salt;
Pyridinium,
4-[2-[4-[(1-oxodecyl)piperazinyl]phenyl]ethenyl]-1(sulfobutyl)-, inner
salt;
Pyridinium,2-[2-[4-[(1-oxooctyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-
inner salt; and
Pyridinium,
2-[2-[4-[(1-oxodecyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt.
Preferred compounds of formula I, of the third aspect of the invention,
include:
Pyridinium, 4-[2-[4-(dibutylamino)phenyl]-1-cyanoethenyl]-1-(sulfobutyl)-,
inner salt; and
Pyridinium, 4-[2-[4-(dihexylamino)phenyl]-1-cyanoethenyl]-1-(sulfobutyl)-,
inner salt.
Compounds of formula I may be prepared from starting materials that are
known by procedures known to those of ordinary skill in the art. By way of
illustration, a synthesis of the compounds of formula I is shown in Scheme
A, below. Compounds of formula Al are disclosed in EP-A-0548798.
Non-limiting examples of the reactions involved may be found in, or based
on, the Examples below. An illustrative synthesis of the compounds of
formula I wherein R.sup.3 is an electron withdrawing substituent as
defined in the third aspect of the invention is shown in Scheme B, below.
##STR4##
##STR5##
Compounds of the invention have utility in the visualisation of proteins
following one-dimensional or 2-dimensional polyacrylamide gel
electrophoresis ("PAGE").
Post-electrophoresis staining with such compounds allows the detection of
proteins, typically down to the level of 2-20 femtomoles. Procedures for
this purpose are known to those of ordinary skill in the art. A preferred
system for 2-dimensional electrophoresis, detection, identification and
isolation of proteins in biological samples is described in U.S.
application Ser. No. 08/980,574, filed Dec. 1, 1997 (published as WO
98/23950), which is incorporated herein by reference in its entirety and
which sets forth a preferred protocol at pages 29-35. In a preferred
embodiment, a biological sample is treated, prior to electrophoresis, to
enrich biomolecules (e.g. proteins) of interest or to deplete biomolecules
(e.g. proteins) that are not of interest, as described in International
Application No. PCT/GB99/01742, filed Jun. 1, 1999, which is incorporated
by reference in its entirety, with particular reference to pages 3 and 6.
For example, a sample (e.g. plasma or serum) may be processed to deplete or
remove one or more proteins such as albumin, haptoglobin, transferrin,
alpha-1-antitrypsin, alpha-2-macroglobulin and immunoglobulin G (IgG) by
performing affinity chromatography whereby the sample is passed through a
series of columns containing immobilized antibodies for selective removal
of albumin, haptoglobin, transferrin, alpha-1-antitrypsin and
alpha-2-macroglobulin, and containing protein G for selective removal of
IgG. In one such embodiment, two affinity columns in a tandem assembly are
prepared by coupling antibodies to protein-G sepharose contained in 1 ml
columns (Protein G-sepharose "Hi-Trap" columns, Pharmacia Cat. No.
17-0404-01) by circulating the following solutions sequentially through
the columns: (1) Dulbecco's Phosphate Buffered Saline (Gibco BRL Cat.
No.14190-094); (2) concentrated antibody solution; (3) 200 mM sodium
carbonate buffer, pH 8.35; (4) cross-linking solution (200 mM sodium
carbonate buffer, pH 8.35, 20 mM dimethylpimelimidate); and (5) 500 mM
ethanolamine, 500 mM NaCl. A third (underivatized) protein G Hi-Trap
column is then attached in series with and following the tandem antibody
column assembly. The chromatographic procedure may be automated using an
Akta Fast Protein Liquid Chromatography (FPLC) System such that a series
of up to seven runs can be performed sequentially. The samples are passed
through the series of 3 Hi-Trap columns, in which the affinity
chromatography media selectively bind the above proteins, thereby
depleting or removing them from the sample. Typically fractions (3 ml per
tube) are collected of unbound material ("flowthrough fractions") that
elutes through the column during column loading and washing, and of bound
proteins ("bound/eluted fractions") that are eluted by step elution with
Immunopure Gentle Ag/Ab Elution Buffer (Pierce Cat. No. 21013). The eluate
containing unbound material is collected in fractions which are pooled,
desalted and concentrated by centrifugal ultrafiltration, and stored to
await further analysis.
A preferred scanner for detecting fluorescently labeled proteins is
described in WO 96/36882 and in the Ph.D. thesis of David A. Basiji,
entitled "Development of a High-throughput Fluorescence Scanner Employing
Internal Reflection Optics and Phase-sensitive Detection (Total Internal
Reflection, Electrophoresis)", University of Washington (1997), Volume
58/12-B of Dissertation Abstracts International, page 6686, the contents
of each of which are incorporated herein by reference. This document
describes a new image scanner designed specifically for automated,
integrated operation at high speeds. The scanner can image gels that have
been stained with fluorescent dyes or silver stains, as well as storage
phosphor screens. The scanner incorporates a phase-sensitive detection
system for discriminating modulated fluorescence from baseline noise due
to laser scatter or homogeneous fluorescence. This capability increases
the sensitivity of the instrument by an order of magnitude or more
compared to conventional fluorescence imaging systems. The increased
sensitivity reduces the sample-preparation load on the upstream
instruments while the enhanced image quality simplifies image analysis
downstream in the process.
5. EXAMPLES
The following illustrative examples are intended to be purely exemplary of
the invention and are not intended to in any way limit its scope.
Example 1
Preparation of N,N-Dipentyl-4-bromo-3-chloroaniline (1).
1-Bromopentane (0.42 mL, 3.4 mmol) was added to a mixture of
4-bromo-3-chloroaniline (200 mg, 0.9 mmol) and potassium carbonate (199
mg, 1.4 mmol) in DMF (20 mL) and the reaction was heated to 13
0.about..about.C overnight. The mixture was cooled, diluted with water (20
mL) and extracted with petroleum ether (3.times.25mL). The combined
organic extracts were dried over sodium sulfate, filtered and concentrated
under reduced pressure. The residue was purified via flash column
chromatography using 9:1 petroleum ether:ethyl acetate as eluent to give
N,N-Dipentyl-4-bromo-3-chloroaniline (1) (332 mg, 68%). .sup.1 HNMR
(CDCI.sub.3) 67.02 (d, IH, ArH); 3.12 (t, 4H, 2.times.NCH.sub.2); 0.95 (t,
6H, 2.times.CH.sub.3). MS; 347 (M+H).
Example 2
Preparation of N,N-Dipentyl-4-bromo-3-methylaniline (2).
Sodium triacetoxyborohydride (1.59 g, 7.5 mmol) was added to a solution of
4-bromo-3-methylaniline (265 mg, 1.4 mmol) and valeraldehyde (8.0 mL, 7.5
mmol) in methanol (25 mL). After stirring for 3 hours the reaction was
quenched with aq. saturated sodium hydrogen carbonate solution (25 mL) and
extracted with petroleum ether (3.times.25mL), The combined organic
extracts were dried over sodium sulfate, filtered and concentrated under
reduced pressure. The residue was purified via flash column chromatography
using petroleum ether as eluent to give
N,N-Dipentyl-4-bromo-3-methylaniline (2) (440 mg, 95%). .sup.1 HNMR
(CDCl.sub.3) 67.14 (d, 1H, ArH). 3.22 (t, 4H, 2.times.NCH.sub.2); 0.82 (t,
6H, 2.times.CH.sub.3). ThC (silica gel): Rf=0.30 (petroleum ether).
Example 3
Preparation of N,N-Dipentyl-4-bromo-3-trifluoromethylaniline (3).
Sodium hydride (480 mg, 1.5 eq.) was added to a solution of
4-bromo-3-trifluoromethylaniline (1.92 g, 5 mmol) and 1-bromopentane (3
mL, 3 eq.) in dimethylformamide (10 mL) at 0.degree. C. After 1 hour a
further (320 mg, 1 eq.) of sodium hydride was added and the reaction was
left overnight. The reaction was heated to 50.degree. C. and left
overnight. The reaction was quenched with methanol and partitioned between
water and petroleum ether. The organic extract was concentrated under
reduced pressure and the residue was purified via flash column
chromatography using petroleum ether as eluent to give
N,N-Dipentyl-4-bromo-3-trifluoromethylaniline (3) (2.84 g. 93%). .sup.1 H
NMIR (CDCl.sub.3) 6 7.34 (d, IH, ArH); 6.80 (d, 1H, ArH); 6.52 (dd, 1H,
ArH); 3.18 (t, 4H, 2.times.NCH.sub.2); 0.86 (t, 6H, CH.about.1b). TLC
(silica gel): RI.about.0.55 (petroleum ether).
Example 4
Preparation of N-Decyl-4-bromo-3-trifluoromethylaniline (4).
Sodium triacetoxyborohydride (4 g) was added to a solution of
3-trifluoromethyl-4-bromoaniline (1.5 g) and decanal (4 mL) in methanol
(90 mL). After stirring for 1 h the solution was partitioned between water
and petroleum ether. The organic extract was concentrated under reduced
pressure and the residue was purified via flash column chromatography
using petroleum ether as eluent to give
N-Decyl-4-broma-3-trifluoromethylaniline (4) (1.2 g, 53%). .sup.1 H NMR
(CDCl.sub.3) .delta. 7.44 (d, 1H, ArH); 6.92 (d, 1H, ArH); 6.61 (dd, 1H,
ArH); 3.11 (t, 2H, NCH.sub.2); 0.80 (t, 3H, CH.sub.2 CH.sub.3). TLC
(silica gel): Rf=0.35 (petroleum ether).
Example 5
Preparation of Intermediate (5)
##STR6##
Palladium diacetate (2 mg, 0.008 mmol) and tri-o-tolylphosphine (24 mg,
0.08 mmol) were added to a solution of
N,N-dipentyl-4-bromo-3-methylaniline (Intermediate 2) (250 mg, 0.77 mmol)
and 4-vinylquinoline (108 mg, 0.70 mmol) in triethylamine (10 mL). The
reaction mixture was heated at reflux for 48 hours. The reaction was
cooled, diluted with water (10 mL) and extracted with dichloromethane
(3.times.20 mL). The combined organic extracts were dried over sodium
sulfate, filtered and concentrated under reduced pressure. The residue was
subjected to flash column chromatography, using 8:2 petroleum ether:ethyl
acetate as eluent, to give Intermediate (5) which was used directly in the
next step.
Example 6
Preparation of Intermediate (6)
##STR7##
Palladium diacetate (9 mg, 0.04 mmol) and tri-o-tolylphosphine (24 mg, 0.04
mmol) were added to a solution of N,N-dipentyl-4-bromo-3-chloroaniline
(Intermediate 1) (120 mg, 0.35 mmol) and 4-vinylquinoline (60 mg, 0.4
mmol) in triethylamine (10 mL). The reaction mixture was heated at reflux
for 48 hours. The reaction was then cooled, diluted with water (10 mL) and
extracted with dichloromethane (3.times.20 mL). The combined organic
extracts were dried over sodium sulfate, filtered and concentrated under
reduced pressure. The residue was subjected to flash column
chromatography, using 8:2 petroleum ether:ethyl acetate as eluent, to give
Intermediate (6) which was used directly in the next step.
Example 7
Preparation of Intermediate (7)
##STR8##
Palladium diacetate (30 mg, ca. 5 mol %) triphenylphosphine (35 mg, ca. 5
mol %) potassium acetate (745 mg, 1 eq.) and tetra-N-butylammonium
chloride (ca. 0.8 g, 1 eq.) were added to a solution of
N,N-dipentyl-4-bromo-3-trifluoromethylaniline (Intermediate 3) (760 mg)
and 4-vinylquinoline (360 mg, 1.1 eq.) in dimethylformamide (5 mL). The
reaction mixture was heated at 110.degree. C. for 4 hours then further
palladium diacetate (30 mg) and triphenylphosphine (35 mg) were added.
After 6 h, tetra-N-butylammonium chloride (0.6 g) was added and the
reaction left overnight. The reaction was then heated to 130.degree. C.
for 4 h, diluted with water (10 mL) and extracted with dichloromethane
(3.times.5mL). The combined organic extracts were dried over sodium
sulfate, filtered and concentrated under reduced pressure. The residue was
purified via flash column chromatography, using a gradient of 0.fwdarw.16%
ethyl acetate:petroleum ether as eluent, to give Intermediate (7) (180 mg
(20%). .sup.1 H NMR (CDCl.sub.3) .delta. 8.72 (d, 1H, ArH); 6.75 (m, 1H,
ArH); 6.54 (dd, 1H, ArH); 3.22 (t, 4H, 2.times.NCH.sub.2); 0.78 (t, 6H,
2.times.CH.sub.2 CH.sub.3). TLC (silica gel): Rf=0.7 (30% ethyl
acetate:petroleum ether).
Example 8
Preparation of Intermediate (8)
Palladium diacetate (35 mg, ca. 5 mol %), triphenylphosphine (88 mg, ca. 10
mol %),
##STR9##
potassium acetate (ca. 1 g, 1 eq.) and tetra-N-butylammonium chloride (ca.
0.9 g, 1 eq.) were added to a solution of
N-decyl-4-bromo-3-trifluoromethylaniline (Intermediate 4) (1.2 g) and
4-vinylquinoline (440 mg, 1 eq.) in wet dimethylformamide (15 mL). The
reaction mixture was heated at 100.degree. C. overnight. Further palladium
diacetate (35 mg), triphenylphosphine (88 mg) and tetra-N-butylammonium
chloride (0.8 g) were added and the reaction mixture heated at 130.degree.
C. for 60 hours. The reaction was diluted with water and extracted with
dichloromethane. The organic extract was dried over sodium sulfate,
filtered and concentrated under reduced pressure. The residue was purified
via flash column chromatography, using a gradient of 10.fwdarw.16% ethyl
acetate: petroleum ether as eluent, to give Intermediate (8) (368 mg,
26%). .sup.1 H NMR (CDCl.sub.3) .delta. 8.69 (d, 1H, ArH); 6.67 (m, 1H,
ArH); 6.58 (m, 1H, ArH); 0.69 (t, 3H, CH.sub.2 CH.sub.3). TLC (silica
gel): Rf=0.55 (30% ethyl acetate:petroleum ether).
Example 9
Preparation of Intermediate (9)
##STR10##
Palladium diacetate (6 mg, ca. 5 mol %) and tri-o-tolylphosphine (15 mg,
ca. 10 mol %) were added to a solution of
N,N-dipentyl-3-trifluoromethyl-4-bromoaniline (Intermediate 3) (190 mg,
0.5 mmol) and 4-vinylpyridine (0.15 mL, 3 eq.) in triethylamine (3 mL) in
a reactivial. The reaction was heated at 110.degree. C. for 5 days. The
residue was purified via flash column chromatography, using a gradient of
20.fwdarw.27.5% ethyl acetate:petroleum ether as eluent, to give
Intermediate (9) (27 mg). .sup.1 H NMR (CDCl.sub.3) .delta. 8.52 (d, 2H,
ArH); 7.68 (d, 1H, ArH); 7.33 (d, 2H, ArH); 3.32 (t, 4H,
2.times.NCH.sub.2); 0.93 (t, 6H, 2.times.CH.sub.2 CH.sub.3). TLC (silica):
Rf=0.70 (50% ethyl acetate:petroleum ether).
Example 10
Preparation of Quinolinium,
4-[2-[4-(dipentylamino)-3-methyl-phenyl]ethenyl]-1-(sulfobutyl)-inner salt
(10)
##STR11##
A mixture of Intermediate (5) (58 mg) and 1,4-butanesultone (2 mL) was
heated at 135.degree. C. for 1 hour. The reaction mixture was diluted with
dichloromethane (3 mL), loaded onto a column of silica and eluted with 1:1
ethyl acetate:ethanol to give quinolinium,
4-[2-[4-(dipentylamino)-3-methyl-phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt (10) (52 mg). .sup.1 H NMR (CDCl.sub.3) .delta. 9.55 (d, 1H, ArH);
5.01 (t, 3H, N+CH.sub.2); 0.78 (t, 6H, 2.times.CH.sub.3). MS 537 (M+H).
Example 11
Preparation of Quinolinium,
4-[2-[4-(dipentylamino)-3-chloro-phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt (11)
##STR12##
A mixture of Intermediate (6) (100 mg, 0.25 mmol) and 1,4-butanesultone (2
mL) was heated at 135.degree. C. for 1 hour. The reaction mixture was
diluted with dichloromethane (3 mL), loaded onto a column of silica and
eluted with 1:1 ethyl acetate:ethanol to give quinolinium,
4-[2-[4-(dipentylamino)-3-chloro-phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt (11) (194mg). .sup.1 H NMR (CDCl.sub.3) .delta. 9.75 (d, 1H, ArH);
5.03 (t, 3H, N(quat)CH.sub.2); 0.77 (t, 6H, 2.times.CH.sub.3). TLC (silica
gel):Rf=0.34 (15% MeOH/dichloromethane)
Example 12
Preparation of Quinolinium,
4-[2-[4-(dipentylamino)-3-trifluoromethyl-phenyl]-thenyl]-1-(sulfobutyl)-,
inner salt (12)
##STR13##
A mixture of Intermediate (7) (232 mg) and 1,4-butanesultone (2mL) were
heated at 135.degree. C. for 1 hour. The reaction mixture was diluted with
dichloromethane (6 mL), loaded onto a column of silica and eluted using a
gradient of 7:1.fwdarw.3:1 dichloromethane:methanol to give quinolinium,
4-[2-[4-(dipentylamino)-3-trifluoromethyl-phenyl]ethenyl]-1-(sulfobutyl)-,
inner salt (12) (324 mg, 64%). .sup.1 H NMR (CD.sub.3 OD) .delta. 9.08 (d,
1H, ArH); 8.85 (dd, 1H, ArH); 4.96 (t, 2H, N.sup.+ CH.sub.2); 0.93 (t, 6H,
2.times.CH.sub.3). MS 613 (M+Na).sup.+ ; 591 (M+H).
Example 13
Preparation of Quinolinium,
4-[2-[4-(decylamino)-3-trifluoromethyl-phenyl]ethenyl]-1-(sulfobutyl)-,
inner salt (13)
##STR14##
A mixture of Intermediate (8) (368 mg) and 1,4-butanesultone (3.5mL) were
heated at 135.degree. C. for 1 hour. The reaction mixture was diluted with
dichloromethane (15mL), loaded onto a column of silica and eluted using a
gradient of 9:1.fwdarw.3.5:1 dichloromethane:methanol to give quinolinium,
4-[2-[4-(decylamino)-3-trifluoromethyl-phenyl]ethenyl]-1-(sulfobutyl)-,
inner salt (13) (344 mg). .sup.1 H NMR (CD.sub.3 OD+CDCl.sub.3) .delta.
9.06 (d, 1H, ArH); 8.83 (m, 1H, ArH); 4.97 (t, 2H, N.sup.+ CH.sub.2); 0.86
(t, 3H, CH.sub.3). MS 613 (M+Na).sup.+ ; 591 (M+H).sup.+.
Example 14
Preparation of Pyridinium,
4-[2-[4-(dipentylamino)-3-trifluoromethyl-phenyl]ethenyl]-1-(sulfobutyl)-,
inner salt (14)
##STR15##
A mixture of Intermediate (9) (27 mg) and 1,4-butanesultone (0.5 mL) were
heated at 130.degree. C. for 1 h. The reaction mixture was diluted with
dichloromethane (3 mL), loaded onto a column of silica and eluted using a
gradient of 7:1.fwdarw.3:1 dichloromethane:methanol to give pyridinium,
4-[2-[4-(dipentylamino)-3-trifluoromethyl-phenyl]ethenyl]-1-(sulfobutyl)-,
inner salt (14) (24 mg). .sup.1 H NMR (CDCl.sub.3) .delta. 8.64 (d, 2H,
ArH); 7.94 (m, 2H, ArH); 4.48 (t, 2H, N.sup.+ CH.sub.2); 3.40 (t, 4H,
2.times.NCH.sub.2); 0.92 (t, 6H, 2.times.CH.sub.3). MS 540 (M).sup.+. TLC
(silica gel):Rf=0.1 (1:9 methanol:dichloromethane).
Example 15
Preparation of Intermediate (15)
##STR16##
To a solution of N-phenylpiperazine (0.76 mL, 5 mmol) and
N,N-diisopropylethylamine (1.74 mL, 2 eq.) in 10 ml of dry dichloromethane
at 0.degree. C., decanoyl chloride (1.25 mL, 1.2 eq.) was added dropwise.
After 0.5 h the reaction mixture was partitioned between water and ether.
The ether fraction was washed with aq. saturated NaHCO.sub.3, dried over
sodium sulfate and concentrated under reduced pressure to a yellow solid.
This material was dissolved in a mixture of diethyl ether and petroleum
ether and concentrated under reduced pressure until a white solid formed
in solution. The white crystals were filtered off and washed with
petroleum ether to give Intermediate (15) (780 mg, 50%). .sup.1 H NMR
(CDCl.sub.3).delta. 7.26 (m, 3H, ArH); 6.97 (m, 2H, ArH); 3.20 (m, 4H,
2.times.NCH2); 0.81 (t, 3H, CH.sub.2 CH.sub.3). TLC (silica gel): Rf=0.45
(40% ethyl acetate:petroleum ether).
Example 16
Preparation of Intermediate (16)
##STR17##
To a solution of N-phenylpiperazine (0.76 mL, 5 mmol) and
N,N-diisopropylethylamine (1.74 mL, 2 eq.) in dry dichloromethane (10 mL)
at 0.degree. C., octanoyl chloride (1.02 mL, 1.2 eq.) was added dropwise.
After lh the reaction was quenched with aq. saturated NaHCO.sub.3. After a
further 15 min the resultant mixture was partitioned between water and
ether. The ether fraction was washed with aq. saturated NaHCO.sub.3, dried
over sodium sulfate and concentrated under reduced pressure to ca. 10 mL.
This concentrate was diluted with petroleum ether (200 mL) and cooled
until white crystals formed. The crystals were filtered off and washed
with petroleum ether to give Intermediate (16) (852 mg, 60%). TLC (silica
gel): Rf=0.6 (60% ethyl acetate:petroleum ether).
Example 17
Preparation of Intermediate (17)
##STR18##
N-bromosuccinimide (440 mg, 1 eq.) was added to a solution of
N-decanoyl-N'-phenylpiperazine (780 mg) in dimethylformamide (20 mL).
After 1 h the reaction mixture was partitioned between water and
dichloromethane. The organic fraction was washed with water, dried over
sodium sulfate and concentrated under reduced pressure. The residue was
purified via flash column chromatography using a gradient of 40.fwdarw.60%
ethyl ether:petroleum ether as eluent to give Intermediate (17) (800 mg,
82%). .sup.1 H NMR .delta.(CDCl.sub.3) .delta. 7.36 (d, 2H, ArH); 6.88 (d,
2H, ArH); 3.20 (m, 4H, 2.times.NCH.sub.2); 0.65 (t, 3H, CH.sub.2
CH.sub.3). TLC (silica gel) Rf0.45 (40% ethyl acetate:petroleum ether).
Example 18
Preparation of Intermediate (18)
##STR19##
N-bromosuccinimide (526 mg, 1 eq.) was added to a solution of
N-octanoyl-N'-phenylpiperazine (852 mg) in dimethylformamide (20 mL).
After 3 h the reaction was partitioned between water and ethyl ether. The
organic fraction was washed with water, dried over sodium sulfate and
concentrated under reduced pressure. The residue was dissolved in ether
(10 mL) and diluted with petroleum ether (200 mL) and then concentrated
under reduced pressure until white crystals were deposited. The white
crystals were filtered off and washed with petroleum ether to give
Intermediate (18) (770 mg, 71%). .sup.1 H NMR (CDCl.sub.3).delta. 7.33 (d,
2H, ArH); 6.87 (m, 2H, ArH); 3.18 (m, 4H,2.times.NCH.sub.2); 0.87 (t, 3H,
CH.sub.2 CH.sub.3). TLC (silica gel): Rf=0.6 (60% ethyl acetate:petroleum
ether).
Example 19
Preparation of Intermediate (19)
##STR20##
Palladium diacetate (7 mg, ca. 5 mol %), triphenylphosphine (17 mg, ca. 10
mol %), potassium acetate (180 mg, 2 eq.) and tetra-N-butylammonium
chloride (210 mg, leq.) were added to a solution of Intermediate (17) (259
mg, 0.65 mmol) and 4-vinylquinoline (101 mg, 1 eq.) in dimethylformamide
(4 mL). The reaction mixture was heated at 105.degree. C. overnight. The
reaction was then diluted with water and extracted with dichloromethane.
The organic extract was dried over sodium sulfate, filtered and
concentrated under reduced pressure. The residue was purified via flash
column chromatography, using a gradient of 40.fwdarw.70% ethyl
acetate:petroleum ether as eluent, to give Intermediate (19). .sup.1 H NMR
(CDCl.sub.3) .delta. 8.81 (d, 1H, ArH); 8.23 (t, 2H, ArH); 6.92 (d, 2H,
ArH); 3.24 (m, 4H, 2.times.NCH.sub.2); 0.84 (t, 3H,CH.sub.2 CH.sub.3). TLC
(silica gel): Rf=0.20 (60% ethyl acetate:petroleum ether).
Example 20
Preparation of Intermediate (20)
##STR21##
Palladium diacetate (11 mg, ca. 5 mol %) and triphenylphosphine (30 mg, ca.
10 mol %) were added to a solution of Intermediate (18) (367 mg, 1 mmol)
and 4-vinylquinoline (155 mg, 1 eq.) in triethylamine (4 mL) in a
reacti-vial. The reaction was heated to 110.degree. C. for 48 hours. The
reaction was then concentrated under reduced pressure. The residue was
purified via flash column chromatography, using a gradient of
40.fwdarw.90% ethyl acetate:petroleum ether as eluent, to give
Intermediate (20) (160 mg, 36%). .sup.1 H NMR (CDCl.sub.3) .delta. 8.89
(d, 1H, ArH); 8.26 (dd, 2H, ArH); 6.98 (d, 2H, ArH); 3.32 (m, 4H,
2.times.NCH.sub.2); 0.92 (t, 3H, CH2CH.sub.3).TLC (silica gel): Rf=0.20
(60% ethyl acetate:petroleum ether.)
Example 21
Preparation of Intermediate (21)
##STR22##
Palladium diacetate (6 mg, ca. 5 mol %) and triphenylphosphine (17 mg, ca.
10 mol %) were added to a solution of Intermediate (18) (200 mg, 0.54
mmol) and 4-vinylpyridine (0.12 mL, 2 eq.) in triethylamine (3 mL) in a
reacti-vial. The reaction mixture was heated at 110.degree. C. for 4 days.
On cooling, the triethylamine solution was transferred from a solid
precipitate and concentrated under reduced pressure. The residue was
purified via flash column chromatography, using a gradient of
30.fwdarw.100% ethyl acetate:petroleum ether as eluent, to give
Intermediate (21) (52 mg). .sup.1 H NMR (CDCl.sub.3).delta. 8.52 (d, 2H,
ArH); 7.43 (d, 2H, ArH); 6.88 (d, 2H, ArH); 3.22 (m, 4H,
2.times.NCH.sub.2); 0.84 (t, 3H, CH.sub.2 CH.sub.3). TLC (silica gel):
Rf=0.10 (60% ethyl acetate:petroleum ether).
Example 22
Preparation of Intermediate (22)
##STR23##
Palladium diacetate (8 mg, ca. 5 mol %) and tri-o-tolylphosphine (21 mg,
ca. 10 mol %) were added to a solution of Intermediate (17) (270 mg, 0.68
mmol) and 4-vinylpyridine (0.15 mL, 2 eq.) in triethylamine (4 mL) in a
reacti-vial. The reaction was heated at 110.degree. C. for 4days. The
reaction was cooled and concentrated under reduced pressure. The residue
was purified via flash column chromatography, using a gradient of
40.fwdarw.100% ethyl acetate:petroleum ether as eluent, to give
Intermediate (22) (130 mg). .sup.1 H NMR (CDCl.sub.3).delta. 8.52 (d, 2H,
ArH); 7.45 (d, 2H, ArH); 6.89 (d, 2H, ArH); 3.22 (m, 4H,
2.times.NCH.sub.2); 0.82 (t, 3H, CH.sub.2 CH.sub.3). TLC (silica
gel):Rf=0. 10 (60% ethyl acetate:petroleum ether).
Example 23
Preparation of Intermediate (23)
##STR24##
Palladium diacetate (6 mg, ca. 5 mol %) and tri-o-tolylphosphine (17 mg,
ca. 10 mol %) were added to a solution of Intermediate (18) (200 mg, 0.54
mmol) and 2-vinylpyridine (0.18mL, 3 eq.) in triethylamine (3 mL) in a
reacti-vial. The reaction was heated at 110.degree. C. for 2days. The
reaction was cooled, diluted with toluene and filtered to remove a white
precipitate. The resultant solution was concentrated under reduced
pressure and the residue was purified via flash column chromatography,
using a gradient of 30.fwdarw.75% ethyl acetate:petroleum ether as eluent,
to give Intermediate (23) (73 mg). .sup.1 H NMR (CDCl.sub.3).delta. 8.68
(d, 2H, ArH); 7.45 (d, 2H, ArH); 6.90 (d, 2H, ArH); 3.22 (m, 4H,
2.times.NCH.sub.2); 0.88 (t, 3H, CH.sub.2 CH.sub.3). TLC (silica
gel):Rf=0.10 (50% ethyl acetate:petroleum ether).
Example 24
Preparation of Intermediate (24)
##STR25##
Palladium diacetate (6 mg, ca. 5 mol %) and tri-o-tolylphosphine (17 mg,
ca. 1 mol %) were added to a solution of Intermediate (17) (200 mg, 0.5
mmol) and 2-vinylpyridine (0.15 mL, 3 eq.) in triethylamine (3 mL) in a
reacti-vial. The reaction was heated at 110.degree. C. for 2days. The
reaction was cooled, diluted with toluene and filtered to remove a white
precipitate. The resultant solution was concentrated under reduced
pressure and the residue was purified via flash column chromatography,
using a gradient of 40.fwdarw.70% ethyl acetate:petroleum ether as eluent,
to give Intermediate (24) (138 mg). .sup.1 H NMR (CDCl.sub.3).delta. 8.57
(d, 2H, ArH); 7.34 (d, 2H, ArH); 6.90 (d, 2H, ArH); 3.22 (m, 4H,
2.times.NCH.sub.2); 0.88 (t, 3H, CH.sub.2 CH.sub.3). TLC (silica
gel):Rf=0.10 (50% ethyl acetate:petroleum ether).
Example 25
Preparation of Quinolinium,
4-[2-[4-[(1-oxodecyl)piperazinyl]phenyl]ethenyl]1-(sulfobutyl)-, inner
salt (25)
##STR26##
A mixture of the total product of Intermediate (19) and 1,4-butanesultone
(2 mL) were heated at 135.degree. C. for 1.5 h. The reaction mixture was
diluted with dichloromethane (6 mL), loaded onto a column of silica and
eluted using a gradient of 7:1.fwdarw.3:1 dichloromethane:methanol to give
quinolinium,
4-[2-[4-[(1-oxodecyl)piperazinyl]-phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt (25) (70 mg). .sup.1 H NMR (CD.sub.3 OD).delta. 8.98 (d, 1H, ArH);
8.81 (m, 1H, ArH); 3.73 (t, 4H, 2.times.NCH.sub.2); 0.84 (t, 3H,
CH.sub.3).MS 605 (M.sup.+)
Example 26
Preparation of Quinolinium,
4-[2-[4-[(1-oxooctyl)piperazinyl]phenyl]ethenyl]1-(sulfobutyl)-, inner
salt (26)
##STR27##
A mixture of Intermediate (20) (160 mg) and 1,4-butanesultone (2.5 mL) were
heated at 130.degree. C. for 1 h. The reaction mixture was diluted with
dichloromethane (8 mL), loaded onto a column of silica and eluted using a
gradient of 7:1.fwdarw.2.5:1 to give dichloromethane:methanol quinolinium,
4-[2-[4-[(1-oxooctyl)-piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt (26) (176 mg). .sup.1 H NMR (CD.sub.3 OD+CD.sub.3 COCD.sub.3) .delta.
9.09 (d, 1H, ArH); 8.90 (m, 1H, ArH); 5.02 (t, 2H, N+CH.sub.2); 3.75
(t,4H, 2.times.NCH.sub.2); 0.88 (t, 3H, CH.sub.3). TLC (silica gel):
Rf=0.10 (10% methanol: dichloromethane).
Example 27
Preparation of Pyridinium,
4-[2-[4-[(1-oxooctyl)piperazinyl]phenyl]ethenyl]-(sulfobutyl)-, inner salt
(27)
##STR28##
A mixture of Intermediate (21) (47 mg) and 1,4-butanesultone (1 mL) were
heated at 130.degree. C. for 1 h. The reaction mixture was diluted with
dichloromethane (5 mL), loaded onto a column of silica and eluted using a
gradient of 7:1.fwdarw.2:1 dichloromethane:methanol to give pyridinium,
4-[2-[4-[(1-oxooctyl)piperazinyl]-phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt (27) (45 mg). .sup.1 H NMR (CD.sub.3 OD) .delta. 8.67 (d, 2H, ArH);
8.04 (d, 2H, ArH); 4.53 (t, 2H, N.sup.+ CH.sub.2); 3.72 (m, 4H,
2.times.NCH.sub.2); 0.92 (m, 3H, CH.sub.3).). MS 527 (M).sup.+. TLC
(silica gel): Rf=0.05 (1:7 methanol: dichloromethane).
Example 28
Preparation of Pyridinium,
4-[2-[4-[(1-oxodecyl)piperazinyl]phenyl]ethenyl]1-(sulfobutyl)-, inner
salt (28)
##STR29##
A mixture of Intermediate (22) (127 mg) and 1,4-butanesultone (2mL) were
heated at 130.degree. C. for 1 h. The reaction mixture was diluted with
dichloromethane (8 mL), loaded onto a column of silica and eluted using a
gradient of 7:1.fwdarw.2:1 dichloromethane:methanol to give pyridinium,
4-[2-[4-[(1-oxooctyl)piperazinyl]-phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt (28) (162 mg). .sup.1 H NMR (CDCL.sub.3) .delta. 8.63 (d, 2H, ArH);
7.98 (d, 2H, ArH); 4.48 (t, 2H, N.sup.+ CH.sub.2); 3.73 (m, 4H,
2.times.NCH.sub.2); 0.87 (m, 3H, CH.sub.3).). MS 555 (M).sup.+. TLC
(silica gel): Rf=0.05 (1:7 methanol: dichloromethane).
Example 29
Preparation of Pyridinium,
2-[2-[4-[(1-oxooctyl)piperazinyl]phenyl]ethenyl]1-(sulfobutyl)-, inner
salt (29)
##STR30##
A mixture of Intermediate (23) (70 mg) and 1,4-butanesultone (1 mL) were
heated at 130.degree. C. for 1 h. The reaction mixture was diluted with
dichloromethane (8 mL), loaded onto a column of silica and eluted using a
gradient of 7:1.fwdarw.3:1 dichloromethane:methanol to give pyridinium,
4-[2-[4-[(1-oxooctyl)piperazinyl]-phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt (29) (41 mg). .sup.1 H NMR (CDCL.sub.3) .delta. 8.72 (d, 1H, ArH);
7.03 (d, 2H, ArH); 4.73 (t, 2H, N.sup.+ CH.sub.2); 3.73 (m, 4H,
2.times.NCH.sub.2); 0.88 (m, 3H, CH.sub.3).). MS 526 (M-H).sup.+.
Example 30
Preparation of Pyridinium,
2-[2-[4-[1(1-oxodecyl)piperazinyl]phenyl]ethenyl] 1-(sulfobutyl)-, inner
salt (30)
##STR31##
A mixture of Intermediate (24) (133 mg) and 1,4-butanesultone (1.5 mL) were
heated at 130.degree. C. for 1 h. The reaction mixture was diluted with
dichloromethane (6 mL), loaded onto a column of silica and eluted using a
gradient of 7:1.fwdarw.3:1 dichloromethane:methanol to give pyridinium,
2-[2-[4-[(1-oxooctyl)piperazinyl]-phenyl]ethenyl]-1-(sulfobutyl)-, inner
salt (30) (110 mg). .sup.1 H NMR (CDCL.sub.3) .delta. 8.72 (d, 1H, ArH);
7.02 (d, 2H, ArH); 4.73 (t, 2H, +CH.sub.2); 3.72 (m, 4H,
2.times.NCH.sub.2); 0.87 (m, 3H, CH.sub.3).). MS 554 (M-H).sup.+. TLC
(silica gel): Rf=0.05 (1:9 methanol: dichloromethane).
Example 31
Preparation of Intermediate (31)
##STR32##
4-Acetonitrilepyridine hydrochloride (465 mg, 3 mmol) was dissolved in 4 ml
of water and neutralized with solid K.sub.2 CO.sub.3 (205 mg). The
solution was partitioned between water and ethyl acetate and the organic
layer washed with water, dried and concentrated to a solid. The solid was
dissolved in a 2 ml of 1,4-butanesultone and stirred at 110.degree. C. for
2 h. The reaction mixture was diluted with acetone and solid precipitated.
The mixture was stirred for 15 mins to grind up the purple solid then
filtered. The solid was washed with acetone and dried to give Intermediate
(31) (514 mg, 67%). .sup.1 H NMR (d.sup.6 -DMSO) .delta. 9.24 (d,2H, ArH);
8.27 (d, 2H, ArH); 4.77 (t, 2H, N.sup.+ CH.sub.2). TLC (silica gel):
Rf=0.45 (1:1 dichloromethane:methanol).
Example 32
Preparation of N,N-Dihexyl-4-aminobenzaldehyde (32).
To a solution of dry DMF (1.1. mL) under Argon at 0.degree. C. was added of
POC13(375 .mu.L, 4 mmol). N,N-Dihexylaniline (1.04 g, 4 mmol) was added
dropwise and the solution was mixed with rapid stirring to form a green
solution. The reaction mixture was heated at 95.degree. C. for 2 h. The
solution quickly turned red/brown. After 2 h the solution was cooled. It
was poured into ice/water 20 mL and aq. saturated NaOAc (20 mL) was added.
The partially neutralized solution was partitioned between ether and
water. The ether layer was washed with aq. saturated NaHCO3 and dried over
sodium sulfate. After concentrating to approximately 20 mL. DMF oiled out
of the solution. The solution was transferred from the oil and
concentrated under reduced pressure. The resultant residue was purified
via flash column chromatography, using a gradient of 0.fwdarw.20% ethyl
acetate; petroleum ether as eluent to give N,N-Dihexyl-4-aminobenzaldehyde
(32) (970 mg, 84%). .sup.1 H NMR (CDCl.sub.3) .delta. 9.72 (s, 1H CHO);
7.70 (d, 2H, ArH); 6.64 (d, 2H, ArH); 3.32 (t, 4H, 2.times.NCH.sub.2);
0.89 (t, 6H 2.times.CH.sub.2 CH.sub.3). TLC (silica gel): Rf=0.75 (20%
ethyl acetate;petroleum ether).
Example 33
Preparation of Pyridinium,
4-[2-[4-(dibutylamino)phenyl]-1-cyano-ethenyl]-1-(sulfobutyl)-, inner salt
(33)
##STR33##
N,N-Dibutyl-4-aminobenzaldehyde (96 mg, 0.41 mmol) was dissolved in ethanol
(1.5 ml) and Intermediate (31) (125 mg, 1.2 eq) was added. A red colour
began to form. Pyrrolidine (50 .mu.L) was added to the solution and the
solid sultone immediately dissolved. After stirring overnight a red solid
had formed and was collected by filtration and dried to give pyridinium,
4-[2-[4-(dibutylamino)phenyl]-1-cyano-ethenyl]-1-(sulfobutyl-, inner salt
(33) (161 mg, 84%). .sup.1 H NMR (d.sup.6 -DMSO) .delta. 8.72 (d, 2H,
ArH); 6.83 (d, 2H, ArH); 4.54 (t, 2H N.sup.+ CH.sub.2); 0.93 (t, 6H,
2.times.CH.sub.2 CH.sub.3). TLC (silica gel): Rf=0.2
(7:1dichloromethane:methanol)
Example 34
Preparation of Pyridinium,
4-[2-[4-(dihexylamino)phenyl]-1-cyano-ethenyl]-1-(sulfobutyl)-, inner salt
(34)
##STR34##
To a solution of Intermediate (32) (159 mg, 1.1. eg.) in ethanol (2 mL) was
added Intermediate (31) (127 mg, 0.5 mmol). A red colour began to form in
the purple suspension. Pyrrolidine (50 .mu.L) was added and the suspension
dissolved to give a dark red solution. After 7 h, a heavy red precipitate
had formed in the flask. This was filtered off; washed with ethanol and
diethyl ether and dried to give pyridinium,
4-[2-[4-(dihexylamino)phenyl]-1-cyano-ethenyl]-1-(sulfobutyl), inner salt
(34) (176 mg, 67%). .sup.1 HMR (CD.sub.3 OD+D.sub.2 O) .delta. 8.73 (d,
2H, ArH); 6.89 (d, 2H, ArH); 4.59 (t, 2H, N.sup.+ CH.sub.2); 0.94 (t, 6H
2.times.CH.sub.2 CH.sub.3). MS 527 (M+H).sup.+.
The present invention is not to be limited in scope by the specific
embodiments disclosed in the examples which are intended as illustrations
of a few aspects of the invention and any embodiments which are
functionally equivalent are within the scope of this invention. Indeed,
various modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art and are
intended to fall within the appended claims.
All references disclosed herein are hereby incorporated by reference in
their entirety.
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